Interferon alpha 2b modified by polyethylene glycol, the preparation and use thereof

ABSTRACT

The present invention relates to interferon-α2b modified with Y-shaped branched polyethylene glycol (PEG) at a single Lys residue and the preparation thereof. The peglated IFN-α2b can be used for the preparation of a medicament for treating a disease, e.g. viral infections such as Hepatitis C.

FIELD OF THE INVENTION

The present invention relates to interferon-α2b modified with Y-shapedbranched polyethylene glycol (PEG) at a single amino acid residue andthe preparation thereof, as well as the use of the prepared PEGylatedIFN-α2b at a single amino acid residue in pharmaceutical field.

BACKGROUND OF THE INVENTION

Interferons (IFNs) are a family of small molecule proteins orglycoproteins produced by eukaryotic cells in response to viralinfection and other antigenic stimuli, which display broad-spectrumantiviral, antiproliferative and immunomodulatory effects. IFNs havebeen widely applied in the treatment of various conditions and diseases,such as viral infections, e.g. hepatitis B, hepatitis C and HIV;inflammatory disorders and diseases, e.g. multiple sclerosis, arthritis,asthma, cystic fibrosis and interstitial lung disease; and tumors, e.g.myelomas, lymphomas, liver cancer, lung cancer, hairy-cell leukemia, andso on (Kenji Oritani, Paul W Kincade, et al. Type I interferon andlimitin: a comparison of structures, receptors, and functions. Cytokineand Growth Factor Reviews, 12, 337-348, 2001; Yu-Sen Wang, StephenYoungster, et al. Structural and biological characterization ofPEGylated recombinant interferon alpha-2b and its therapeuticimplications. Advance Drug Delivery Reviews, 54, 547-570, 2002).

IFNs are classified into four types according to their differences inchemical, immunological, and biological properties: interferon-α, β, γand ε. Interferon-α (IFN-α) is secreted by leukocytes. Human IFNs-α areencoded by a multigene family consisting of about 20 genes, the encodedproteins sharing up to about 90% amino acid sequence homology (Henco K.,Brosius F. J., et al. J. Mol. Biol., 185, 227-260, 1985). Human IFN-α2bis one of the subtypes of the α2 subfamily of human IFN-α family, and isa single chain protein with various biological activities. The singlechain protein consists of 165 amino acid residues with 4 Cys, whereintwo intrachain disulfide bonds are formed between Cys1-Cys98 andCys29-Cys138 respectively and the N-terminal amino acid is Cys with onefree α-NH₂ group. The residues in positions 31, 49, 70, 83, 112, 121,131, 133, 134 and 164 of the amino acid sequence are Lys, each of whichcontains one free ε-NH₂ group. This protein is not glycosylated andsensitive to many proteases. The amino acid sequence of humaninterferon-α2b is shown in SEQ ID No: 1.

IFNs are usually administered parenterally in clinical treatments. Theshort in vivo half-life (2-4 h) and strong immunogenicity of IFNs resultin a shorter dosing interval and a higher dosing frequency. As thegenerated antibodies significantly decrease the therapeutic efficacy, itis difficult to achieve ideal clinical efficacy. The polyethylene glycol(PEG) modification technology developed in recent years has provided apossible solution to the above problems.

PEG is an inert, nontoxic and biodegradable organic polymer, and isimportant in the fields of both biotechnology and pharmaceutics. PEGmodification technique is to link PEG to an active protein via covalentbond. After the polyethylene-glycolation (PEGylation), the properties ofthe protein can be significantly improved, e.g. the prolongation of drugmetabolic half-life, the reduction of immunogenicity, the increase ofsafety, the improvement of therapeutic efficacy, the decrease of dosingfrequency, the increase of drug solubility/water solubility, theincrease of resistance against proteolysis, the facilitation of drugcontrolled release and so on. For further details please refer to Inadaet al. J. Bioact. and Compatible Polymers, 5, 343, 1990, Delgado et al.Critical Reviews in Therapeutic Drug Carrier Systems, 9, 249, 1992,Katre. Advanced Drug Delivery Systems, 10, 91, 1993, and U.S. Pat. No.4,179,337.

It is disclosed in U.S. Pat. No. 4,179,337, after linking PEG to anenzyme or insulin, the immunogenicity of the protein was reduced, whilesimultaneously the activities of the protein were reduced as well. Thiswas also found in G-CSF (Satake-Ishikawa et al. Cell Structure andFunction, 17, 157-160, 1992), IL-2 (Katre et al. Proc. Natl. Acad. Sci.USA, 84, 1487, 1987), TNF-α (Tsutsumi et al. Jpn. J. Cancer Res., 85, 9,1994), IL-6 (Inoue et al. J. Lab. Clin. Med., 124, 529, 1994) andCD4-IgG (Chamow et al. Bioconj. Chem., 5, 133, 1994).

It was reported that the branched PEG-modified protein exhibited betterpH tolerance, thermo-stability and resistance against proteolysis thanlinear chain PEG-modified proteins (Monfardini et al. BioconjugateChem., 6, 62, 1995). Generally, a PEG molecule modifies a protein bylinking itself to the N-terminal α-amino group or ε-amino groups of aLys residue within the protein molecule. There are normally three typesof PEGs for protein modification: a linear chain PEG (EP 0593868), anU-shaped branched PEG (EP 0809996) and an Y-shaped branched PEG(CN1243779C).

Currently many kinds of PEGylated proteins have been applied clinically.In 1990, the PEGylated-bovine adenosine deaminase (Adagen) produced byENZON Inc. was approved by FDA, and used to treat severe combinedimmunodeficiency disease (pegfamg013102LB, http://www.fda.gov). In 1994,another PEG-modified protein for treating acute lymphoblastic leukemia,the PEGylated asparaginase (pegaspargase, Oncaspar), was also marketedin US (103411s5052lbl, http://www.fda.gov). The PEG modifiedinterferon-α2b (PEG IFN-α2b, PEG-Intron) developed by Schering-Ploughwas approved by FDA for marketing in 2000 and the PEGylated interferon-α(PEG IFN-α2a, Pegasys) produced by Hoffman-la Roche Ltd. was alsoapproved for marketing in 2002, both of which are used to treathepatitis (103964s5037lbl, pegsche011901LB, http://www.fda.gov). In2002, the PEG modified human granulocyte colony-stimulating factorproduced by Amgen Inc. (PEG-filgrastim, Neulasta) was also approved byFDA, which is used to treat metastatic breast cancer (pegfamg013102LB,http://www.fda.gov). The FDA also accepted the application for PEGylatedhuman growth factor antagonist developed by Pharmacia. The PEG combinedTNF-α antibody fragment from Celltech and the PEG-TNF receptor fromAmgen are tested in the advanced clinical trials. The first PEG-organicmolecule conjugate, PEGylated camptothecin, has also entered phase II ofclinical trial. In 2004, the PEG modified oligonucleotide (Pegaptanib,Macugen™) was approved by FDA. The in vivo metabolism of the PEG in thedrug (or PEG itself) has already been clearly understood, and PEG hasbeen proven to be a good and safe drug modifier without any adverseeffect.

The PEGs that can be linked to a protein drug normally needderivatization, so that one or two terminal groups of the ends of PEGscan be chemically activated to possess a proper functional group whichdisplays activity, and thus can form a stable covalent bond with, atleast one functional group of the drug to be linked. For example, PEGscan be linked to ε-NH₂ of Lys residue within the protein peptide chain,or to α-NH₂ of the N-terminal amino acid residue of the protein peptidechain. In the PEGylation of IFN-α described in European patentEP0809996, PEG-NHS is linked through nucleophilic substitution to α-NH₂of the N-terminal amino acid or ε-NH₂ of Lys in IFN-α. The PEG-NHSmentioned in the above patent is a U-shaped branched PEG derivative(PEG₂-NHS), the molecular formula thereof as below:

wherein, R and R′ are independently a low molecular weight alkyl group,n and n′ are from 600 to 1500, and the average molecular weight of thePEG is from 26 KD to 66 KD. The molecular formula of thePEG₂-NHS-modified IFN-α is as below:

Where one or more PEG₂-NHS molecules are linked to α-NH₂ of theN-terminal amino acid or ε-NH₂ of Lys in IFN-α, the obtained productsare a mixture of non-PEGylated IFNs-α, PEGylated IFNs-α at a singleamino acid residue and PEGylated IFNs-α at multiple amino acid residues.The PEGylated IFN-α at a single amino acid residue can be isolated fromthe obtained products by any appropriate purification means. IFN-α hasone N-terminal amino acid and more than one Lys residues, namely severalreactive sites for PEG₂-NHS, so the isolated PEGylated IFNs-α at asingle amino acid residue are a mixture of the isomers of the PEGylatedIFNs-α at different single amino acid residues.

In European patent EP 0593868, linear-chain PEG is used to modify IFN,the molecular formula of the modified product as below:

wherein R is a low molecular weight alkyl group; R₁, R₂, R₃ and R₄ are Hor low molecular weight alkyl groups; m is from 1 to the number ofpossible PEG modification positions in IFN; W is O or NH; x is from 1 to1000, y and z are from 0 to 1000, x+y+z is from 3 to 1000; and at leastone of R₁, R₂, R₃ and R₄ is a low molecular weight alkyl group. Yu-SenWang et al (Yu-Sen Wang et al, Advanced Drug Delivery Reviews, 54:547-570, 2002. Yu-Sen Wang et al, Biochemistry, 39, 10634-10640, 2000.)have reported the modification of rIFN-α2b with 12 KD linearmonomethoxy-PEG (Peg-Intron) and shown that the products analyzed byHPLC-IE are a mixture of more than 14 isomers modified by PEG atdifferent single amino acid residues. The molecular formula of thelinear PEGs used by Yu-Sen Wang et al is shown below:

wherein the average molecular weight of the PEG is 12 KD.

SUMMARY OF THE INVENTION

The PEG derivatives used in the present invention are novel branched,Y-shaped branched PEG derivatives, and their structures are differentfrom those of the U-shaped branched PEGs. The biggest difference betweenthese two kinds of PEGs is that: two-branch PEG chains of the Y-shapedPEG derivatives according to the present invention are connectedtogether through N atom, while the two-branch PEG chains of the U-shapedPEG derivatives in EP0809996 are connected together through C atom. Themolecular composition of the Y-shaped PEG derivatives according to thepresent invention is shown as below:

wherein, P_(a) and P_(b) are same or different PEGs; j is an integerfrom 1 to 12; R_(i) is H, a substituted or unsubstituted C1-C12 alkylgroup, a substituted aryl, an aralkyl or a heteroalkyl; X₁ and X₂ areindependently a linking group, wherein X₁ is (CH₂)_(n), and X₂ isselected from the group consisting of (CH₂)_(n), (CH₂)_(n)OCO,(CH₂)_(n)NHCO, and (CH₂)_(n)CO; n is an integer from 1 to 10; and F is aterminal group selected from the group consisting of a hydroxyl group, acarboxyl group, an ester group, acyl chloride, hydrazide, maleimide,pyridine disulfide, capable of reacting with amino, hydroxyl or mercaptogroup of a therapeutic agent or a substrate to form a covalent bond.

In one preferred embodiment of the present invention, the Y-shaped PEGderivative molecule is shown as below:

wherein, R and R′ are independently a C1-C4 alkyl group, preferablymethyl; m and m′ denote the degree of polymerization and can be anyinteger; m+m′ is preferably from 600 to 1500; R_(i) is H, a substitutedor unsubstituted C1-C12 alkyl, a substituted aryl, an aralkyl, or aheteroalkyl group; j is an integer from 1 to 12; and F is a terminalgroup selected from the group consisting of a hydroxyl group, a carboxylgroup, an ester group, carboxylic acid chloride, hydrazide, maleimide,pyridine disulfide, capable of reacting with an amino group, a hydroxylgroup or a mercapto group of a therapeutic agent or a substrate to forma covalent bond. Preferably, the average total molecular weight of thePEG is from about 10000 to about 60000 Dalton, most preferably about40000 Dalton.

In one preferred embodiment of the present invention, a possiblestructural formula of the Y-shaped PEG derivative molecule is shown asformula (I):

wherein R and R′ are independently a C1-C4 alkyl group, preferablymethyl; m and m′ denote the degree of polymerization and can be anyinteger; m+m′ is preferably from 600 to 1500; j is an integer from 1 to12; and the average total molecular weight of the PEGs is about 40000Dalton.

The present inventors used Y-shaped branched PEG derivatives (YPEG) tomodify interferon-α2b (IFN-α2b), and isolated the YPEG-IFNs-α2b,modified by PEG at a single amino acid residue, by Q-Sepharose FFion-exchange chromatography. Moreover, the isolated YPEG-IFNs-α2b,modified by PEG at a single amino acid residue, were further separatedby SP-Sepharose FF chromatography to obtain YPEG-IFN-α2b wherein theYPEG is principally linked to the side chain ε-NH₂ of Lys at position134 in SEQ ID NO.1, which is called YPEG-IFN-α2b(134). Aftermeasurement, it is found that the in vitro activity of theYPEG-IFN-α2b(134) is significantly higher than that of the YPEG-IFN-α2bin which the YPEG is linked to another amino acid residue, and thehalf-life of the YPEG-IFN-α2b(134) in serum is significantly longer thanthat of the unmodified IFN-α2b.

Therefore, the present invention provides PEGylated IFNs-α2b at a singleamino acid residue, the structure of which is as below:

wherein P_(a) and P_(b) are same or different PEGs; j is an integer from1 to 12; R_(i) is H, a substituted or unsubstituted C1-C12 alkyl group,a substituted aryl, an aralkyl, or a heteroalkyl group; X₁ and X₂ areindependently a linking group, wherein X₁ is (CH₂)_(n), and X₂ isselected from the group consisting of (CH₂)_(n), (CH₂)_(n)OCO,(CH₂)_(n)NHCO and (CH₂)_(n)CO, wherein n is an integer from 1 to 10.

In one preferred embodiment of the present invention, the PEGylatedIFNs-α2b of the present invention is of the structural formula (II)below:

wherein R and R′ are independently a C1-C4 alkyl group, preferablymethyl; j is an integer from 1 to 12; m and m′ denote the degree ofpolymerization and can be any same or different integers. In said thisstructure, a Y-shaped branched PEG molecule is linked to an IFN-α2bmolecule via one single amino acid residue. The average molecular weightof the YPEG-IFN-α2b in formula (II) depends principally on the degree ofpolymerization, m and m′. Where m+m′ is preferably from 600 to 1500, thecorresponding average molecular weight of the YPEG is from about 26000to about 66000 Dalton. Where m+m′ is preferably from 795 to 1030, thecorresponding average molecular weight of the YPEG is from about 35000to about 45000 Dalton. Where m+m′ is preferably from 885 to 1030, thecorresponding average molecular weight of the YPEG is from about 39000to about 45000 Dalton. Where m+m′ is most preferably 910, thecorresponding average molecular weight of the YPEG is 40000 Dalton. Theratio of m and m′ can be in a range from 0.5 to 1.5, preferably from 0.8to 1.2.

In one preferred embodiment, in the PEGylated IFN-α2b of the presentinvention, a PEG molecule is linked to IFN-α2b via an amido bond formedby α-amino group of the N-terminal amino acid or the side chain ε-aminogroup of Lys residue of IFN-α2b corresponding to position 31, 49, 70,83, 112, 121, 131, 133, 134, or 164 as shown in SEQ ID No.1.

In a further preferred embodiment, in the PEGylated IFN-α2b of thepresent invention, a PEG molecule is linked to IFN-α2b via an amido bondprincipally formed by the side chain ε-amino group of Lys residue ofIFN-α2b corresponding to position 134 as shown in SEQ ID No.1.

Optionally, the IFN-α2b of the present invention can be extracted fromnatural sources or obtained by the recombinant biotechnology.Preferably, the IFN-α2b is human IFN-α2b (hIFN-α2b) having the aminoacid sequence of SEQ ID No.1, which is extracted from natural sources orobtained by the recombinant biotechnology. More preferably, the humanIFN-α2b is recombinant human IFN-α2b (rhIFN-α2b). The rhIFN-α2b can beartificially synthesized, or be expressed from prokaryotic expressionsystems such as E. coli, or be expressed from eukaryotic yeastexpression systems such as Pichia, or be expressed from insect cellexpression systems or mammalian cell expression systems such as CHO. Thepreparation methods of the natural or recombinant IFN-α2b and theactivity tests of IFN-α2b and YPEG modified IFN-α2b are known in priorart.

Similar to IFN-α2b, the YPEG-IFN-α2b of the present invention can alsobe used clinically to treat tumors and viral infections, such ashepatitis, hairy-cell leukemia, cell-mediated lympholysis, Kaposi'ssarcoma and so on. In clinical, the YPEG-IFN-α2b of the presentinvention is clearly improved, as compared to IFN-α2b, in stability,solubility, half-life in serum and clinical therapeutic efficacy. Forthe mode of administration, the YPEG-IFN-α2b of the present inventioncan be administered to the patients in the form of a compositioncomprising a pharmaceutically effective amount of the YPEG-IFN-α2b and apharmaceutically acceptable carrier or excipient. Hence, the presentinvention, in another aspect, also provides a composition comprising apharmaceutically effective amount of the PEGylated IFN-α2b of thepresent invention and a pharmaceutically acceptable carrier orexcipient. Preferably, the composition comprises mannitol, amino acids,sodium chloride and sodium acetate, wherein the amino acids arepreferably selected from the group consisting of aspartic acid,asparagine and glycine.

In another aspect, the present invention also provides the use of thePEGylated IFN-α2b of the invention or the composition comprising thePEGylated IFN-α2b of the invention in the preparation of a medicamentfor treating a disease in need of IFN-α2b treatment. Preferably, thedisease in need of IFN-α2b treatment is selected from the groupconsisting of viral infections e.g. hepatitis B, hepatitis C, hepatitisD and condyloma acuminatum, tumors e.g. hairy-cell leukemia, chronicmyeloid leukemia, low-grade malignant non Hodgkin's leukemia,cell-mediated lympholysis, Kaposi's sarcoma, multiple myeloma, malignantmelanoma, cutaneous T-cell lymphoma, laryngeal papilloma, recurrent ormetastatic renal cell carcinoma, inflammatory disorders and diseasese.g. multiple sclerosis, arthritis, asthma, cystic fibrosis andinterstitial lung disease, and myeloproliferative diseases relatedthrombocythemia.

In order to obtain the YPEG modified IFN-α2b, in one embodiment of thepresent invention, initially the PEG moiety of activated YPEGderivatives such as PEG N-hydroxyl succinimidyl ester (YPEG-NHS) iscovalently linked to an amino (—NH₂) group of the protein throughnucleophilic substitution, wherein the amino group includes N-terminalα-amino group of the protein and an ε-amino group of Lys residue. Thereaction equation for the generation of YPEG-IFN-α2b from IFN-α2b andYPEG is as below:

The reaction conditions are mild, the pH is in a range from 4.5 to 9.5,the temperature is between 0-25° C., and stirring or other blendingmeasures are necessary. For detailed conditions please refer to theExamples in DETAILED DESCRIPTION OF THE INVENTION. All YPEGs withdifferent molecular weights can be linked to IFN-α2b using the abovemethod. The products include the modified by PEG at a single amino acidresidue (YPEG-IFN-α2b), the modified by PEG at two amino acid residues(YPEG₂-IFN-α2b) and the modified by PEG at multiple amino acid residues(YPEG_(n)-IFN-α2b), wherein the products modified by PEG at a singleamino acid residue can be the predominant products by adjusting thereaction condition.

Subsequently, the YPEG-IFN-α2b, modified by PEG at a single amino acidresidue, can be isolated from the mixture of all kinds of the YPEGmodified IFNs-α2b using a method such as cation exchange chromatography,anion exchange chromatography, or exclusion chromatography, and then theIFNs-α2b modified by PEG at different single amino acid residues can befurther resolved to obtain the YPEG-IFN-α2b in which the YPEG is linkedat a specific position. Conventional purification methods include cationexchange chromatography, anion exchange chromatography, hydrophobicinteraction chromatography and exclusion chromatography. Characteristicanalysis can be performed by a known method in the art, e.g. the massspectroscopy, the polyacrylamide gel electrophoresis and thehigh-performance liquid exclusion chromatography can be used to analyzethe molecular weight of the products, so as to distinguish the productsmodified by PEG at a single amino acid residue from those modified byPEG at two or multiple amino acid residues and unmodified IFN-α2b. Theabove mentioned purification methods can also be used to further resolvethe products modified by PEG at a single amino acid residue to obtaindifferent isomers with the PEG modification at different singlepositions. The in vitro biological activities of all kinds of the PEGmodified products can be measured according to any known assay forIFN-activity, e.g. cytopathic effect inhibition. For IFNs modified byPEG at a single amino acid residue, the PEG moieties in the differentisomers have different effects on maintaining the active domains ofIFNs, resulting in the great differences in the biological activities ofdifferent isomers. Generally speaking, the in vitro activities of IFNsare remarkably decreased after PEG modification. However, according tothe present invention, the in vitro specific activity of the isolates ofthree peaks obtained by ion exchange chromatography have been measured,and the results indicate that the isolate of peak 3 (SP2) hassignificantly higher specific activity than the isolates of other peaksand PEGASYS (Hoffmann-La Roche, Basel, Switzerland), and hassignificantly longer half-life in serum than unmodified IFN-α2b.

In a further embodiment, the Y-branched PEG linked peptide isolated fromthe SP2 is sequenced using Edman degradation, and the results showedthat the primary component of SP2 was YPEG-IFN-α2b(134).

Hence, in another aspect, the present invention also provides thepreparation and purification methods for YPEG-IFN-α2b(134), comprising:

(a) under an alkaline condition, preferably at pH 9.0, allowing Y-shapedbranched PEG as shown in formula (I) below to react with IFN-α2b, andobtaining PEGylated IFN-α2b;

wherein R and R′ are independently a C1-C4 alkyl group, preferablymethyl; j is an integer from 1 to 12; m and m′ denote the degree ofpolymerization and can be any integer; and m+m′ is preferably from 600to 1500;

(b) capturing the reaction products in step (a) with an anion exchangeresin, preferably Q Sepharose FF, and eluting the products in an aniongradient, preferably in a chloride ion gradient, to obtain modifiedproducts;

(c) eluting the reaction products captured in step (b) with a cationexchange resin, preferably SP Sepharose FF, in a cation gradient,preferably in a sodium ion gradient, and collecting each peakseparately;

(d) determining the activity of the product from each peak, andselecting the peak corresponding to the reaction product with highestactivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: SDS-PAGE of 2 batches of IFN-α2b modified with YPEG (40 KD). Theconcentration of the separation gel was 12%, and Coomassie brilliantblue R-250 was used as staining dye. It can be seen from the SDS-PAGEresult of FIG. 1, under the condition, the PEG modification rate ofrHuIFN-α2b was between 30-50%, and was stable. The primary modifiedproducts were IFN-α2b modified by PEG at a single amino acid residue(YPEG-IFN-α2b), and there were also some IFN-α2b modified by PEG atmultiple amino acid residues (YPEGn-IFN-α2b). Lane 1: IFN-α2b, NHS-YPEG(40 KD) modification reaction at 0 h; Lane 2: Batch 060604-1 of IFN-α2b,NHS-YPEG (40 KD) modification reaction at 2 h; Lane 3: Batch 060604-2 ofIFN-α2b, NHS-YPEG (40 KD) modification reaction at 2 h; Lane 4: marker(GE Lifescience).

FIG. 2: The resolving profile of YPEG-IFN-α2b modification isomers bySP-Sepharose FF.

FIG. 3: Silver-stained SDS-PAGE (12%) of the YPEG-IFN-α2b samplespurified by SP-Sepharose FR Lane 1: molecular weight marker (GELifescience); Lane 2: SP-Sepharose FF purification peak 1 ofYPEG-IFN-α2b; Lane 3: SP-Sepharose FF purification peak 2 ofYPEG-IFN-α2b, Lane 4: SP-Sepharose FF purification peak 3 ofYPEG-IFN-α2b; Lane 5: SP-Sepharose FF purification peak 4 ofYPEG-IFN-α2b.

FIG. 4: Apparent molecular weight of the SP-Sepharose FF purifiedYPEG-rHuIFN-α2b sample determined by 7.5% reducing SDS-PAGE with silverstaining. Lane 1: molecular weight marker (GE Lifesciences); Lane 2:YPEG-HuIFN-α2b SP1, 2 μg; Lane 3: YPEG-rHuIFN-α2b SP2, 2 μg; Lane 4:YPEG-rHuIFN-α2b SP3, 2 μg.

FIG. 5: The molecular weights of the SP-Sepharose FF purifiedYPEG-IFN-α2b samples determined by MALDI-TOF MS.

FIG. 6: The molecular weight of YPEG-NHS (40 KD) determined by MALDI-TOFMS.

FIG. 7: The serum drug concentration and activity after a single s.c.injection of 30 μg·kg⁻¹ YPEG-rhIFN-α2b into Crab-eating Macaque (Macacafascicularis).

FIG. 8: The blank control of Trypsinase Peptide Mapping of the trypsindigested YPEG-rHuIFN-α2b SP2. Two small peaks were detected respectivelyat 71.674 min and 17.589 min; and the trypsin peak was detected between2-3 min.

FIG. 9: The analysis of Trypsinase Peptide Mapping of the trypsindigested (0 h) YPEG-IFN-α2b SP2 sample by HPLC-RP C₁₈. The retentiontime of YPEG-IFN-α2b SP2 was 62.114 min; and the elution peak at 71.908min was the background, 2-3 min.

FIG. 10: The analysis of Trypsinase Peptide Mapping of the trypsindigested (48 h) YPEG-IFN-α2b SP2 sample by HPLC-RP C₁₈. No substratepeak (62.114 min) was detected between 60.5 min-63.2 min, demonstratingthat the sample was completely digested.

FIG. 11: Sephacryl S-100 HR separation profile of the YPEG modifiedpeptides from the trypsin completely digested YPEG-IFN-α2b SP2 sample.The samples were collected according to the peaks, S100-1 was YPEGmodified peptides, namely the target sample.

DETAILED DESCRIPTION OF THE INVENTION

The present invention will be further described by the followingexamples, but any example or the combination thereof should not beunderstood as limiting the scope or embodiment of the invention. Thescope of the invention is limited only by the appended claims. Incombination with the description and prior art, skilled persons in theart would clearly understand the scope limited by the claims.

Example 1 Preparation of Y-shaped Branched PEG Modified RecombinantHuman IFN-α2b (1) Small-scale Preparation of Y-shaped Branched PEGModified Recombinant Human IFN-α2b

166.1 mg of YPEG (formula (I), average molecular weight 40 KD,equal-arm, lot number RD010P041, Beijing JenKem Technology Co., Ltd.)was weighted and dissolved in 1 ml of 2 mM HCl (Guangdong GuanghuaChemical Factory Co., Ltd.). And 40 mg IFN-α2b (Xiamen Amoytop BiotechCo., Ltd.) and 50 mM boric acid-borax buffer (pH 9.0, Sinopharm ShanghaiChemical Reagent Co., Ltd.) were added to a final reaction volume of 10ml. In this reaction system, the final concentration of IFN-α2b was 4mg/ml, and the reaction molar ratio of IFN-α2b and YPEG was 1:2. Thereaction system was kept under 0-20° C. for 2 h with stirring. ThePEGylated IFNs-α2b were then generated, and the reaction was stopped byadding glacial acetic acid (Shantou Xilong Chemical Co., Ltd.) to makepH<4.0. A sample was subjected for SDS-PAGE. The reaction system wasdiluted 50 times with water and then 0.2 μm filtered before stored at 4°C. for further use.

Q-Sepharose FF Chromatography was used to separate the remaining PEG andPEG hydrolates, IFNs-α2b modified by YPEG at multiple amino acidresidues, IFNs-α2b modified by YPEG at a single amino acid residue andthe unmodified IFN-α2b. Q-Sepharose FF (GE Healthcare) column (Φ12 mm×90mm, 1 CV=10 ml) was regenerated with 3 CV of 20 mM boric acid/boraxbuffer (pH9.0)-1 M NaCl (BBI), and then equilibrated with 5 CV of 20 mMboric acid/borax buffer (pH9.0). UV detection wavelength was set at 280nm. The entire sample stored at 4° C. was loaded. After loading, thecolumn was equilibrated with 3 CV of boric acid/borax buffer (pH9.0),and then 20 mM boric acid/borax buffer (pH9.0)-12 mM NaCl was used forelution until the first peak was completely eluted, which peak was theremaining PEG. 20 mM boric acid/borax buffer (pH9.0)-60 mM NaCl was thenused for elution, and the sample collected in this elution peak wasprimarily the YPEG-IFNs-α2b, modified by PEG at a single amino acidresidue. And then 20 mM boric acid/borax buffer (pH9.0)-500 mM NaCl wasused for elution and the elution peak was the unmodified IFN-α2b.

The target products were primarily the YPEG-IFNs-α2b modified by PEG ata single amino acid residue, with a yield rate of 20-40%.

2) Large-scale Preparation of Y-shaped Branched PEG Modified RecombinantHuman IFN-α2b

4982.4 mg of YPEG (formula (I), average molecular weight 40 KD,equal-arm, lot number RD010P041, Beijing JenKem Technology Co., Ltd.)was weighted and dissolved in 25 ml of 2 mM HCl. And 1200 mg IFN-α2b and50 mM boric acid/borax buffer (pH 9.0) were added to a final reactionvolume of 200 ml. In this reaction system, the final reactionconcentration of IFN-α2b was 6 mg/ml, and the reaction molar ratio ofIFN-α2b and YPEG was 1:2. The reaction system was kept under 0-25° C.for 2 h with stirring. The reaction was stopped by adding glacial aceticacid to make pH<4.0. A sample was subjected for SDS-PAGE. The reactionsystem was diluted 50 times with water and then 0.2 μm filtered beforestored at 4° C. for further use.

Q-Sepharose FF Chromatography was used to separate the remaining PEG andPEG hydrolates, IFNs-α2b modified by YPEG at multiple amino acidresidues, IFNs-α2b modified by YPEG at a single amino acid residue andthe unmodified IFN-α2b. Q-Sepharose FF (GE Healthcare) column (Φ38mm×265 mm, 1 CV=300 ml) was regenerated with 3 CV of 20 mM boricacid/borax buffer (pH9.0)-1 M NaCl, and then equilibrated with 5 CV of20 mM boric acid/borax buffer (pH9.0). UV detection wavelength was setat 280 nm. The entire sample stored at 4° C. was loaded. After loading,the column was equilibrated with 3 CV of boric acid/borax buffer(pH9.0), and then 20 mM boric acid/borax buffer (pH9.0)-12 mM NaCl wasused for elution until the first peak was completely eluted, which peakwas the remaining PEG. 20 mM boric acid/borax buffer (pH9.0)-60 mM NaClwas then used for elution, and the sample collected in this elution peakwas primarily the YPEG-IFNs-α2b modified by PEG at a single amino acidresidue. And then 20 mM boric acid/borax buffer (pH9.0)-500 mM NaCl wasused for elution and the elution peak was the unmodified IFN-α2b.

The target products were primarily the YPEG-IFNs-α2b modified by PEG ata single amino acid residue, with a yield rate of 35-50%.

FIG. 1 shows SDS-PAGE results for 2 batches of IFNs-α2b modified withYPEG (40 KD). It can be seen from FIG. 1 that under the condition, thePEG modification rate of rhIFN-α2b was between 35-50% and remainedstable. The primary modified products were modified by PEG at a singleamino acid residue (YPEG-IFN-α2b), and there were also some productsmodified by PEG at multiple amino acid residues (YPEG_(n)-IFN-α2b).

Example 2 Resolving YPEG-IFNs-α2b by SP-Sepharose FF

The Q-Sepharose FF captured YPEG-IFNs-α2b sample was adjusted to pH 5.0with 20% acetic acid, then diluted 15 times with 5 mM NaAc/HAc (pH5.0,Shantou Xilong Chemical Co., Ltd.). The sample was loaded at 0.5 mg/mlloading capacity to SP-Sepharose FF 100 ml (GE Healthcare) column (Φ18mm×394 mm). The column was equilibrated with 3 CV of 5 mM NaAc/HAc(pH5.0), and then eluted with 2.5 CV of the gradient of 0%-30% 5 mMNaAc/HAc-70 mM NaCl (pH5.0), following with 50 CV of the gradient of30%-100% 5 mM NaAc/HAc-70 mM NaCl (pH5.0). YPEG-IFN-α2b was resolved as4 elution peaks by SP-Sepharose FF 100 ml. The samples were collectedaccording to these peaks and then measured by SDS-PAGE with silverstaining respectively. According to the SDS-PAGE results, it can be seenthat peak 1 resolved by SP-Sepharose FF was primarily the productsmodified by YPEG at multiple amino acid residues (YPEG_(n)-IFN-α2b).Peak 2 by SP-Sepharose FF was primarily the products modified by PEG ata single amino acid residue (YPEG-IFN-α2b), and also contained someproducts modified by PEG at multiple amino acid residues. Peak 3 andpeak 4 by SP-Sepharose FF were both the products modified by PEG at asingle amino acid residue. Peaks 2-4 resolved by SP-Sepharose FF wereisomers with YPEG-modification at different single positions, and werenamed respectively as YPEG-IFN-α2b SP1, YPEG-IFN-α2b SP2 andYPEG-IFN-α2b SP3. The resolution profile and silver-stained SAD-PAGEresults were shown in FIG. 2 and FIG. 3 respectively.

Every sample of YPEG-IFN-α2b SP1-3 was supplemented with sodiumchloride, sodium acetate, mannitol, aspartic acid and was sterilizedwith 0.22 μm filter before stored at 4° C. for further use.

Example 3 Characteristic Analysis of YPEG-IFN-α2b Modification Isomers(1) Protein Concentration

The concentrations of YPEG-IFN-α2b modification isomers were determinedby Kjeldahl method.

(2) Protein Apparent Molecular Weight

The apparent molecular weights of YPEG-IFN-α2b modification isomers weredetermined by SDS-PAGE. The method was according to Laemmli et al(Nature 227: 680, 1970). The concentration of the gel was 7.5%, and thegel was visualized by silver staining. The apparent molecular weights ofYPEG-IFN-α2b modification isomers were almost the same, about 123 KD(FIG. 4).

(3) Molecular Weight Determined by MALDI-TOF MS

MALDI-TOF MS (Autoflex TOF/TOF system, Bruker Daltonics, Germany) wasused to determine the molecular weights of YPEG-rHuIFN-α2b modificationisomers. Sinapinic acid (SA, C₁₁H₁₂O₅, M.W. 224.22, lot number: 2006236870 002, Bruker Daltonics, Germany) was used as matrix. ProteinCalibration Standard H (Part No. 207234, Bruker Daltonics, Germany) wasused as protein molecular weight standard, and the software for dataanalysis was FlexAnalysis Ver. 3.0.54.0. The MS molecular weights ofYPEG-IFN-α2b modification isomers were almost the same, about 59000Dalton (FIG. 5).

(4) Endotoxin Content Test

Based on limulus assay (Pharmacopoeia of the People's Republic of China,2005, Volume 3, Appendix X C), the endotoxin content of everyYPEG-IFN-α2b sample was less than 5.0 EU/mg.

(5) In Vivo Activity and Pharmacokinetics of YPEG-IFN-α2b SP2 in Animal.

{circle around (1)} In Vivo Activity of YPEG-IFN-α2b SP2 in Animal.

The action mechanism of IFN is partially to induce the production of2′,5′-AS (2′,5′-oligoadenylate synthetase), which in turn exerts itsantiviral effects. Using ¹²⁵I as a tracer, the pharmacodynamicparameters of IFN are reflected by the in vivo 2′,5′-AS activity.2′,5′-AS catalyzes the synthesis of 2′,5′-A (2′,5′-oligoadenylate) fromATP in the presence of Poly(I).Poly(C) agar (The activity of 2′,5′-AScan be represented by the concentration of the synthesized 2′,5′-A).First, 2′,5′-AS in the samples are absorbed and activated byPoly(I).Poly(C) agarose, then catalyzes the substrate ATP to generate2′,5′-A. A mixture of ¹²⁵I labeled 2′,5′-A, anti-2′,5′-A serum andsecondary antibody is added into the sample which then is incubated andcentrifugated to separate the mixture. The supernatant is discarded anda Gamma Counter was used to measure the radioactivity of the sediment.The binding rate of the initially added ¹²⁵I labeled 2′,5′-A iscalculated. Four-parameter Logistic regression is used to generatestandard curve, and then the concentration of the 2′,5′-AS-induced2′,5′-A products in an unknown sample could be estimated.

Using the above mentioned 2′,5′-A method, the results in Table 1 andFIG. 7 showed the serum 2′,5′-A concentration after a single s.c.injection of 30 μg·kg⁻¹ YPEG-IFN-α2b SP2 into Crab-eating Macaque(Macaca fascicularis) (18 Crab-eating Macaques, Guangxi WeimeiBiotechnology Co., Ltd., Certification No. S.CXK GUI 2002-0001, bodyweight 2.5-5.5 kg, 6 female and 12 male, raised in separate cages, fedwith standard monkey feed, drink freely). It can be seen from FIG. 8that the average time-to-peak was 64±27.71 h, and the concentration topeak was 292.30±148.08 Pmol·dL⁻¹. After administration, the activity of2′,5′-AS in serum was clearly increased, and the time-to-peak of 2′,5′-Ain serum was delayed than that of YPEG-rhIFNα2b SP2.

TABLE 1 The serum 2′,5′-A concentrations over time, after a single s.c.injection of 30 μg•kg⁻¹ YPEG-rhIFN-α2b SP2 into Crab-eating Macaque.(Pmol · dL⁻¹) No. of crab-eating Macaque time(h) 1 2 3 Mean SD   0 19.32  13.63  20.74 17.90 ± 3.76   1  40.17 218.67 — 129.42 ± 126.22  2  45.74  14.30  80.23 46.76 ± 32.98   4  14.89  69.41 138.23 74.18 ±61.81   8  49.12 243.43 141.66 144.74 ± 97.19  10 119.51 274.99 109.89168.13 ± 92.67  12  72.75 152.81 112.87 112.81 ± 40.03  24  10.05 321.23159.12 163.47 ± 155.63  48  45.60 622.42 164.49 277.50 ± 304.56  96400.67 352.65 123.58 292.30 ± 148.08 168  10.87 286.38   4.17 100.47 ±161.03 240   2.74 323.83  10.48 112.35 ± 183.19 312  20.65 238.65   1.5486.94 ± 131.72 {circle around (2)} Pharmacokinetics of YPEG-IFNs-α2b andrhIFN-α2b in Crab-eating Macaque

A single s.c. injection of 10, 30 or 100 μg·kg⁻¹ YPEG-IFN-α2b was givento Crab-eating Macaque. For the administration group, 1 ml of venousblood was taken from the hind leg opposite to the injected side at thetime before, 1 h, 2 h, 4 h, 8 h, 10 h, 12 h, 24 h, 48 h, 72 h, 96 h, 168h, 240 h, and 312 h after administration. For the group with a singles.c. injection of IFN-α2b (30 μg·kg⁻¹), 1 ml of blood was taken at thetime before, 0.5 h, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 8 h, and 24 h afteradministration. After kept at 4° C. for 30 min, the blood samples werecentrifugated at 2000 rpm for 10 min under low temperature, then theserum was separated immediately and stored at −20° C. for furtheranalysis.

The quantitative double sandwich immunoassay was used. A monoclonalantibody specific to the recombinant human IFN-α was pre-coated onmicrotiter plate. The standard and the samples were pipetted into themicrotiter wells, wherein the IFN-α2b or YPEG-IFN-α2b SP2 would bind tothe immobilized antibody. The plate was washed to remove unboundsubstances, and then anti-human IFN-α IgG (secondary antibody) was addedinto the wells. After the reaction was complete, the plate was washedand the horseradish peroxidase (HRP) was added into the wells. Afterwashing away the unbound enzyme and reagents, the color generated byadding HRP substrate solution into each well was proportional to theamount of the bound IFN-α2b or YPEG-rhIFN-α2b SP2 in the first step. Thereaction was stopped and the color intensity was measured. The higherthe OD value of absorbance, the higher the concentration of IFN-α2b orYPEG-IFN-α2b SP2 in the sample. The standard curves were plotted forIFN-α2b and YPEG-IFN-α2b SP2 respectively so as to measure the serumdrug concentration in the blood samples.

According to the protocol in the description of the kit (AmericanBiomedical Co., lot number 3271), 100 μl standard or blood sample wasadded into each well, and mixed with plate mixer gently. According tothe anticipated concentration of an unknown sample, the sample wasdiluted with the dilute solution to the concentration ranges of thestandard curve. The IFN-α2b or YPEG-IFN-α2b SP2 standard curve for eachplate was plotted so as to calculate the concentration of the unknownsample in that plate. The plate was incubated at room temperature for 1h, and washed once with plate washing solution. 100 μl secondaryantibody was added to each well, and kept under room temperature for 1h. The plate was washed 3 times, and 100 μl HRP conjugate was added toeach well. The plate was incubated under room temperature for 1 h andwashed 4 times. 100 μl TMB substrate was added into each well, and keptunder room temperature in the dark for 15 min. 100 μl stop solution wasadded to each well, and mixed gently to stop the reaction. Theabsorbance OD value at 450 nm was measured with a microplate readerwithin 5 min to determine the concentration of each sample.

After a single subcutaneous injection of low-dose, medium-dose, orhigh-dose (10, 30 and 100 μg·kg⁻¹, respectively) of YPEG-IFN-α2b intoCrab-eating Macaque, the half-lives were 48.87±11.67, 51.94±3.52 and49.60±2.97 h, respectively. After a single subcutaneous injection of 30IFN-α2b into Crab-eating Macaque, the half-life was 3.22±0.10 h. Thehalf-life of IFN-α2b was prolonged at least ten times after YPEGmodification.

(6) The in vitro biological activity of each YPEG-IFN-α2b modificationisomers was estimated using cytopathic effect inhibition assay.According to the method described in Determination Method of InterferonActivity (Pharmacopoeia of the People's Republic of China, 2005, Volume3, Appendix X C), interferon protects human amniotic cells (WISH) fromthe damage caused by vesicular stomatitis virus (VSV). Crystal violetwas used to stain survived WISH cells, and the absorbance OD value wasmeasured at 570 nm. The interferon protection effect curve was plottedfor WISH cells, so as to determine the in vitro biological activity ofinterferons. The results of in vitro biological activity of each samplesare shown in Table 2, and 3 parallel tests were carried out for eachsample. After YPEG modification, in all the modification isomers of theproducts modified by PEG at a single amino acid residue, the SP2 sampleshowed the highest in vitro specific activity, which was 1-2 timeshigher than SP1, SP3 and PEGASYS (manufactured by Hoffmann-La Roche,Basel, Switzerland; packaged separately by Shanghai RochePharmaceuticals Ltd., product lot number B1016, package lot numberSH0020), and was also 1-2 times higher than the unresolved sample.

TABLE 2 In vitro biological activity results for each modificationisomer of YPEG-IFN-α2b (3 parallel tests) Average PEG No. of SpecificM.W. Modification Activity Sample PEG Type (KD) Positions (×10⁶ IU/mg)YPEG-IFN-α2b SP1 Y-branched 40 1  1.07 ± 0.172 YPEG-IFN-α2b SP2Y-branched 40 1  2.65 ± 0.185 YPEG-IFN-α2b SP3 Y-branched 40 1  1.13 ±0.215 YPEG-IFN-α2b Y-branched 40 1  1.68 ± 0.217 unresolved samplePEGASYS U-branched 40 1 0.934 ± 0.042 Note: unresolved sample refers tothe sample before resolving YPEG-rhIFN-α2b by SP-Sepharose FF

(7) The Resolution of the Modification Position in YPEG-IFN-α2b SP2

The solvent system of YPEG-IFN-α2b SP2 was changed to 50 mM NH₄HCO₃(pH8.0) by ultrafiltration with 5K ultrafilter (Millipore,polyethersulfone material), and the protein concentration was determinedto be 3.82 mg/ml using UV spectroscopy. TPCK Trypsin (Promega) wasdissolved (0.5 μg/μl) in the solution provided by the manufacturer.Samples were added according to Table 3:

TABLE 3 Reaction composition of YPEG-IFN-α2b SP2 trypsin digestionReaction Composition Volume 50 mM NH₄HCO3, pH8.0 7.08 ml PEG-IFN-α2b SP2(3.82 mg/ml) 1.32 ml Trypsin (0.5 μg/μl)  0.2 ml Total reaction volume 8.6 ml

The reaction system was kept in a water bath at 37° C. for 48 h, then1.52 ml of 20% acetic acid was added to stop the reaction. A smallamount of sample was taken for HPLC-RP C18 peptide mapping. Theinstrument for analysis was Waters HPLC system, with a controller oftype 600, 2487 double wavelength detector, and the software for dataprocessing was Empower 2. The HPLC analytical column was Jupiter C18(particle diameter 5 μm, pore diameter 300 Å, Φ4.6×150 mm, produced byPhenomenex, USA). Mobile phase A was 0.1% TFA/H₂O, Mobile phase B was0.1% TFA/90% ACN/H₂O, the flow rate was 1 ml/min, and the detectionwavelength was set at 214 nm. Please refer to Table 4 for the elutiongradients, and the results were shown in FIG. 8-10.

TABLE 4 The elution gradients for HPLC-RP C18 peptide mapping of thetrypsin digested YPEG-IFN-α2b SP 2 Time (min) A % B % ACN % 1  0 100  0 0 2  8 100  0  0 3 68  40 60 54 4 72  40 60 54 5 75 100  0  0 6 80 100 0  0

Based on the detection result, it can be determined that the sample wasalmost completely digested. The products were treated with DTT reductionafter the reaction was stopped. The Sephacryl S-100 HR column (Φ18×255mm, 1 CV=64 ml; GE Healthcare) was pre-equilibrated with 3 CV of 20 mMPBNa-400 mM NaCl (pH7.0), and 3% CV of the YPEG-IFN-α2b SP2 sample byTPCK trypsin digested completely was loaded by hydrostatic pressure. 20mM PBNa-400 mM NaCl (pH7.0) was used for elution, and the detectionwavelength was set at 280 nm. The sample from the first elution peak wascollected (sample number: YPEG-IFN-α2b S100-1, FIG. 11), and the solventsystem was changed to 5 mM PBNa (pH 7.0) with 5K ultrafilter. Vacuumfreeze-drying was done. The N-terminal amino acids of the freeze-driedsample were determined using Edman degradation, and the sequence of the7 amino acids at the N-terminus of the sample was XYSPXAW (Table 5),wherein X denotes α-amino acid cysteine (Cys), a non-α-amino acid oranother modified amino acid that cannot be detected using Edmandegradation. According to the sequence shown in SEQ ID NO: 1, it can bedetermined that the YPEG-IFN-α2b SP2 was primarily the products modifiedwith YPEG at Lys134.

TABLE 5 Sequencing result for the N-terminal amino acids of YPEG-IFN-α2bS100-1 The corresponding Detected N-terminal PEG modification SampleSequence position. YPEG-IFN-α2b S100-1 XYSPXAW Lys134 Note: X denotesα-amino acid cysteine a non-α-amino acid or another modified amino acidthat cannot be detected using Edman degradation.

1. A PEGylated interferon-α2b (IFN-α2b) of the structure as below,obtained by linking IFN-α2b with a Y-shaped branched polyethylene glycol(YPEG):

wherein, P_(a) and P_(b) are same or different polyethylene glycol(PEG); j is an integer between 1-12; R_(i) is H, substituted orunsubstituted C1-C12 alkyl group, substituted aryl, aralkyl, orheteroalkyl; and X₁ and X₂ are independently a linking group, wherein X₁is (CH₂)_(n), and X₂ is selected from the group consisting of (CH₂)_(n),(CH₂)_(n)OCO, (CH₂)_(n) NHCO, and (CH₂)_(n) CO, wherein n is an integerbetween 1-10.
 2. The PEGylated IFN-α2b of claim 1, with the structure asbelow

wherein R and R′ are independently a C1-C4 alkyl group, preferablymethyl; j is an integer between 1-12; m and m′ denote the degree ofpolymerization and can be any integer; m+m′ is preferably from 600 to1500, and the average total molecular weight of the YPEG is preferablyfrom about 10000 to about 60000 Dalton, most preferably about 40000Dalton.
 3. The PEGylated IFN-α2b of claim 2, wherein the YPEG is linkedto IFN-α2b via an amido bond formed by α-amino group of the N-terminalamino acid of IFN-α2b or the side chain ε-amino group of a Lys residuewithin IFN-α2b corresponding to position 31, 49, 70, 83, 112, 121, 131,133, 134, or 164 in SEQ ID No.1, preferably position 134 in SEQ ID No.1.4. The PEGylated IFN-α2b of claim 2, wherein the IFN-α2b is extractedfrom a natural source or obtained through recombinant biotechnology,preferably is human IFN-α2b having the amino acid sequence as shown inSEQ ID No.1, most preferably is a recombinant human IFN-α2b.
 5. ThePEGylated IFN-α2b of claim 4, wherein the recombinant human IFN-α2b isartificially synthesized or expressed from an expression system selectedfrom the group consisting of a prokaryotic expression system such as E.coil, an eukaryotic yeast expression system such as Pichia, an insectcell expression system or a mammalian cell expression system such asCHO.
 6. A composition comprising a pharmaceutically effective amount ofthe PEGylated IFN-α2b of any one of claims 1-5 and 12 and apharmaceutically acceptable carrier or excipient.
 7. The composition ofclaim 6, further comprising mannitol, an amino acid, sodium chloride andsodium acetate, wherein the amino acid is preferably selected from thegroup consisting of aspartic acid, asparagine and glycine.
 8. (canceled)9. A method for preparing and purifying the PEGylated IFN-α2b of any oneof claims 1-5 and 12, comprising the steps: (a) under an alkalinecondition, preferably at pH 9.0, allowing Y-shaped branched PEG of thefollowing formula to react with IFN-α2b, and obtaining PEGylatedIFN-α2b;

 wherein R and R′ are independently a C1-C4 alkyl group, preferablymethyl; j is an integer between 1-12; m and m′ denote the degree ofpolymerization and can be any integer, and m+m′ is preferably from 600to 1500; (b) capturing the reaction products obtained in step (a) withan anion exchange resin, preferably Q Sepharose FF, and eluting theproducts in an anion gradient, preferably in a chloride ion gradient, toobtain modified products; (c) eluting the reaction products captured instep (b) with a cation exchange resin, preferably SP Sepharose FF, in acation gradient, preferably in a sodium ion gradient, and thencollecting each peak separately: (d) determining the activity of theproduct from each peak, and selecting the peak corresponding to thereaction product with highest activity.
 10. The method of claim 9,wherein the YPEG has a molecular weight of 40 KG, and preferably is anequal-arm Y-PEG, and more preferably the reaction molar ratio of IFN-α2band YPEG is 1:2.
 11. A method of treating a patient suffered from adisease in need of IFN-α2b treatment, comprising administrating atherapeutically effective amount of the PEGylated IFN-α2b of any one ofclaims 1-6 and 12 or the composition of any one of claims 6-7 to thepatient, wherein the disease in need of IFN-α2b treatment is preferablyselected from the group consisting of viral infections e.g. hepatitis B,hepatitis C, hepatitis D and condyloma acuminatum, tumors e.g.hairy-cell leukemia, chronic myeloid leukemia, low-grade malignant nonHodgkin's leukemia, cell-mediated lympholysis, Kaposi's sarcoma,multiple myeloma, malignant melanoma, cutaneous T-cell lymphoma,laryngeal papilloma, recurrent or metastatic renal cell carcinoma andmyeloproliferative diseases related thrombocythemia, inflammatorydisorders and diseases e.g. multiple sclerosis, arthritis, asthma,cystic fibrosis and interstitial lung disease.
 12. The PEGylated IFN-α2aof claim 3, wherein the YPEG is an equal-arm YPEG of the molecularweight of 40000 Dalton.